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Emerging Agricultural Biotechnologies for Sustainable Agriculture and Food

Security

Article  in  Journal of Agricultural and Food Chemistry · January 2016

DOI: 10.1021/acs.jafc.5b04543

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pubs.acs.org/JAFC

Emerging Agricultural Biotechnologies for Sustainable Agriculture

and Food Security
Jennifer A. Anderson,*,† Martijn Gipmans,‡ Susan Hurst,§ Raymond Layton,† Narender Nehra,#
John Pickett,⊥ Dilip M. Shah,Δ Thiago Lívio P. O. Souza,⊗ and Leena TripathiΠ
†
DuPont Pioneer, Johnston, Iowa 50131, United States
‡
BASF Bioscience Research, c/o metanomics GmbH, Tegeler Weg 33, 10589 Berlin, Germany
§
Arcadia Biosciences, Seattle, Washington 98119, United States
#
Institute for International Crop Improvement, Donald Danforth Plant Science Center, St. Louis, Missouri 63132, United States
⊥
Rothamsted Research, Harpenden, Herts AL5 2JQ, United Kingdom
Δ
Donald Danforth Plant Science Center, St. Louis, Missouri 63132, United States
⊗
Embrapa Arroz e Feijão, Rod. GO-462, km 12, Santo Antônio de Goiás, GO 75.375-000, Brazil
Π
International Institute of Tropical Agriculture (IITA), Nairobi, Kenya

ABSTRACT: As global populations continue to increase, agricultural productivity will be challenged to keep pace without
overtaxing important environmental resources. A dynamic and integrated approach will be required to solve global food
insecurity and position agriculture on a trajectory toward sustainability. Genetically modified (GM) crops enhanced through
modern biotechnology represent an important set of tools that can promote sustainable agriculture and improve food security.
Several emerging biotechnology approaches were discussed in a recent symposium organized at the 13th IUPAC International
Congress of Pesticide Chemistry meeting in San Francisco, CA, USA. This paper summarizes the innovative research and several
of the new and emerging technologies within the field of agricultural biotechnology that were presented during the symposium.
This discussion highlights how agricultural biotechnology fits within the context of sustainable agriculture and improved food
security and can be used in support of further development and adoption of beneficial GM crops.
KEYWORDS: agricultural biotechnology, genetically modified crops, sustainability, food security, semiochemicals

■ INTRODUCTION
Producing enough food, feed, fiber, and biofuel to meet society’s
life”.3 Although food security in a global context is complex and
hinges on many socioeconomic, infrastructural, and political
needs has been the goal of agriculture for thousands of years. factors, the agricultural sector’s ability to maximize crop yield and
Sustainable agriculture attempts to fulfill these same basic production without compromising the environmental resource
societal demands with an emphasis on preserving environmental base will also be important. Agricultural biotechnology offers
quality, decreasing agricultural inputs, reducing environmental great potential for promoting sustainable agriculture and
effects, and sustaining economic viability.1 Over the next 30 improving food security; however, the role and potential
years, the world population is projected to increase to contributions of agricultural biotechnology in the context of
approximately 9 billion people, and the increased demand for sustainability and food security are only beginning to be realized.
food, feed, fiber, and biofuel will challenge the agricultural sector The Convention on Biological Diversity (CBD) defines
to keep pace.1 In addition to increasing population pressure, biotechnology as “any technological application that uses biological
climate change and associated extreme weather events, as well as systems, living organisms, or derivatives thereof, to make or modif y
limitations on the amount of arable land and water that is products or processes for specif ic use”.4 Under this broad definition,
devoted to agriculture, will affect agricultural productivity. agriculture has been using biotechnology to select for desired
Without the development of crops that can outperform current traits, improve germplasm, and enhance crop genetics through
varieties, more land will need to be converted to cultivated acres, selective breeding for thousands of years. More modern genetic
and more inputs will be needed to support increasing societal and molecular tools, including tissue culture, embryo rescue,
demands. For example, Tilman et al. estimated that an additional double haploids, and marker-assisted breeding, also fall under the
∼1 billion hectares (ha) of cultivated land will be needed by 2050 umbrella of agricultural biotechnology and give additional
if current agricultural trends continue.2 Achieving food security context and refinement to this definition. These biotechnology
in the face of these challenges, without increasing the tools help facilitate the development of pure genetic lines and
environmental footprint, will require an integrated and
diversified approach. Received: September 16, 2015
Food security exists “when all people, at all times, have physical Revised: November 20, 2015
and economic access to suf f icient, safe and nutritious food to meet Accepted: November 24, 2015
their dietary needs and food preferences for an active and healthy Published: January 11, 2016

© 2016 American Chemical Society 383 DOI: 10.1021/acs.jafc.5b04543

J. Agric. Food Chem. 2016, 64, 383−393
Journal of Agricultural and Food Chemistry Perspective

Figure 1. Golden mosaic disease symptom in field trials comparing genetically modified dry bean “carioca” seeded advanced lines developed by Embrapa
to be resistant to bean golden mosaic virus (BGMV) (right panels) with their respective conventional recurrent parents (left panels).

have increased the speed and efficiency of selecting desired traits,

relative to traditional breeding. Additionally, genetic and
■ GENETICALLY MODIFIED VIRUS-RESISTANT
COMMON BEAN
molecular tools are being used to produce transgenic (also Common bean (Phaseolus vulgaris L.) is an economically,
referred to as genetically modified (GM)) crops, which are nutritionally, and socially important crop, especially in
central to the field of modern agricultural biotechnology. developing countries in Latin America and eastern and southern
In 2014, over 181 million hectares of GM crops were grown Africa. Brazil is the main producer and consumer country of P.
globally.5 On the basis of current trends, the adoption rate of GM vulgaris. The dry bean is a very popular and relevant crop,
crops with biotech traits is expected to continue to increase, representing the major source of dietary protein. The Brazilian
especially in developing countries.6 To date, most of the per capita consumption can be as high as 17 kg/person/year. The
advancements in genetically modified crops have been focused total growing area in 2013 was 1.89 million hectares, with a mean
on a few key row crops (e.g., cotton, maize, and soybean) that productivity of 1350 kg/ha.7 One of several factors that
express insect-resistant proteins from Bacillus thuringiensis or that compromise common bean yield worldwide is the high number
are tolerant to glyphosate and glufosinate−ammonium herbi- of destructive pathogens that attack the crop and cause serious
cides. Alternative technologies offering tolerance to different damage. Among them is the bean golden mosaic virus (BGMV),
herbicides (e.g., dicamba or 2,4-D), as well as a variety of stacked a geminivirus transmitted by the whitefly (Bemisia tabaci).
traits offering herbicide tolerance in combination with multiple BGMV causes severe foliar yellow mosaic symptoms, stunted
modes of action for insect resistance (e.g., Agrisure or SmartStax) growth, deformation of pods and grains, and abortion of flowers.
are also becoming readily available to farmers. Further In Brazil, losses in grain yield may range from 40 to 100%, and at
advancements in the field are increasing not only the variety of least 200,000 ha of Brazilian farmland is unusable for bean
crops being modified (e.g., vegetable crops, wheat, rice, cowpea, growth due to BGMV. Annual losses ranging between 90,000
and banana) but also the range of genetic modifications being and 280,000 tons of grain would be enough to feed 6−20 million
advanced (e.g., enhanced nutrition, modified composition, adult Brazilians.8
Effective resistance to BGMV controlled by major genes has
improved digestibility, abiotic stress tolerance, virus, bacterial,
not been identified in P. vulgaris germplasm, despite over 40 years
and fungal resistance).
of conventional breeding efforts in Brazil. Insecticide spraying to
Several emerging biotechnology approaches were discussed in
control the whitefly and prevent virus incidence has been
a recent symposium organized at the 13th IUPAC International overused, with limited success and serious environmental
Congress of Pesticide Chemistry meeting in San Francisco, CA, concerns. For this reason, the Brazilian Agricultural Research
USA. The goal of this paper is to highlight advancements in the Corporation (Embrapa) developed a GM dry bean resistant to
field of agricultural biotechnology within the context of the BGMV using a pathogen-derived resistance approach, based
sustainable agriculture and food security. Examples of emerging on RNAi technology.9,10 Biosafety assessment assays managed
traits developed to help improve yield, reduce inputs, and by Embrapa, but with the support of more than 10 different
provide protection from viral, bacterial, and fungal pathogens and partner institutions, have already been performed according to
insect pests, as well as enhance crop performance and the rules established by the National Technical Commission on
productivity under abiotic stress conditions, are discussed. Biosafety (CTNBio, Brazilian Government). The resulting
Additionally, the utility of using a life cycle assessment model biosafety dossier was submitted for evaluation in December
to assess the benefits and potential impacts of agricultural 2010 and the GM virus resistant bean was approved for
biotechnology is also highlighted. commercial growth in Brazil in September 2011.
384 DOI: 10.1021/acs.jafc.5b04543
J. Agric. Food Chem. 2016, 64, 383−393
Journal of Agricultural and Food Chemistry Perspective

Figure 2. Amino acid sequences of MsDef1 and MtDef4. The consensus γ-core motif is shown. Eight conserved cysteines are shown in red.

“Carioca” seeded GM common bean advanced lines derived and MtDef4, from Medicago sativa and Medicago truncatula,
from Pérola and BRS Pontal cultivars have been developed.11 respectively, are structurally similar, but share only 41% amino
Although not grown in other parts of the world, “carioca” seeded acid identity (Figure 2).
beans are the most consumed market class in Brazil, representing Several plant defensins exhibit potent antifungal activity in
around 70% of the internal market. Field trials to evaluate the vitro at micromolar concentrations (Figure 3)16 but display
agronomic performance of the GM virus-resistant advanced different modes of action (MOA). Because of their cationic
lines, which are required for the registration of new cultivars in character, defensins are able to interact with negatively charged
Brazil, have been conducted since 2012 in a national assay molecules present at the fungal cell membrane causing an
network coordinated by Embrapa. The seed production process increase in membrane permeability that leads to cell leakage and
(breeder seed and prebasic seed) also began in 2012. All of these death.16−18 They also induce Ca2+ influx and K+ efflux and
aforementioned activities have been developed following a disrupt a Ca2+ gradient essential for polar growth of hyphal
Stewardship Program specifically created for the Embrapa GM tips.19,20 Some defensins bind with high affinity to specific
virus-resistant bean. sphingolipids present in the fungal cell wall and/or plasma
Partial results of the final field trials have demonstrated that no membrane of their target fungi.21,22 Alternatively, other defensins
grain yield penalty has been observed when the GM virus- are internalized by fungal cells and target key components of
resistant advanced lines are compared with their respective fungal cell processes.23−25 The Shah Laboratory at the Danforth
recurrent parents. In addition, GM lines are resistant to BGMV Center has been investigating structure−activity relationships
under field conditions, presenting no visible symptoms, whereas and MOA of MsDef1 and MtDef4. Although both defensins
BGMV severity ranging from 50 to 70% has been observed in the permeabilize the plasma membrane of Fusarium graminearum,17
recurrent parents and control cultivars (Figure 1). At the they exhibit different MOA. MsDef1 interacts with the cell wall
moment, there is no technical concern about agronomic associated sphingolipid glucosylceramide, and this interaction is
performance or BGMV resistance effectiveness of the GM required for its antifungal activity. MtDef4, on the other hand, is
advanced lines. Other modern “carioca” seeded dry bean internalized into fungal cells and binds strongly to phosphatidic
cultivars developed by Embrapa are also being converted with acid (PA), a key phospholipid second messenger in the cell.25
the event conferring resistance to BGMV. After the conclusion of The γ-core motif is a major determinant of the antifungal activity
all final field trials, the superior virus-resistant line will be in vitro of MtDef4 (Figure 3) and plays a major role in enabling
commercially released in the Brazilian domestic market, PA binding and fungal cell entry of MtDef4.25 Studies are
representing the first GM common bean cultivar in the world. underway to understand the role this interaction plays in the
The development of common bean GM cultivars resistant to MOA of MtDef4.
BGMV will play an important role for the integrated manage- Because of their potent antifungal activity, defensins are being
ment of the viruses transmitted by the whitefly and for food exploited in agricultural biotechnology applications to generate
security in Brazil. disease-resistant crops. Constitutive overexpression of several

■ MECHANISMS OF ACTION OF ANTIFUNGAL PLANT

DEFENSINS AND GENETIC ENGINEERING FOR
plant defensins significantly enhances resistance to fungal and
oomycete pathogens in various transgenic crops.16,18,26−29 For
example, expression of the apoplast-targeted MtDef4 in
DISEASE RESISTANCE transgenic wheat confers strong resistance to an obligate
Fungal and oomycete pathogens impose major constraints biotroph, Puccinia tritici, which causes leaf rust disease (Kaur
globally on agricultural production and food safety. An estimated and Shah, manuscript in preparation). Nonetheless, the
10−15% of crop yields is lost due to these pathogens, despite the challenge is to achieve durable and robust resistance to fungal
continued release of resistant cultivars and chemical fungicides. and oomycete pathogens in transgenic plants through expression
Safe and effective antifungal agents with novel fungus-specific of these proteins without compromising important agronomic
mechanisms of action are urgently needed in agriculture. Plants traits and crop yields.30 Several strategies have emerged to
express various cationic antimicrobial peptides that play a major overcome that challenge. Precise temporal and spatial expression
role in plant defense against fungal and oomycete pathogens. of defensins in plants using pathogen-inducible or tissue-specific
Plant defensins are cysteine-rich antimicrobial peptides of 45−54 promoters can minimize any potential deleterious effects of
amino acids. They are constitutively expressed or induced in constitutive expression. Tailoring the expression of antifungal
response to various biotic or abiotic stresses and are targeted to defensins to match the unique lifestyle of each fungal or
the apoplast or vacuole. They display a conserved tertiary oomycete pathogen can result in a more effective control of the
structure characterized by the presence of one α-helix and three disease.31 Moreover, coexpression of defensins and other
antiparallel β-strands. The α-helix is connected to the second β- antifungal proteins can provide better protection from fungal
strand through a cysteine-stabilized α-helix/β-sheet (α/β) and oomycete infection.32
motif.12−15 The amino acid sequences of these defensins contain In conclusion, with a better understanding of the structure−
a signature γ-core motif GXCX3−9C (where X is any amino acid) activity relationships and MOA of plant defensins, and the
that is conserved among all antimicrobial peptides with disulfide availability of adequate gene expression tools, robust and durable
bonds.16 Despite their structural similarity, primary amino acid resistance to fungal and oomycete pathogens in transgenic crops
sequences of plant defensins exhibit very low sequence is achievable. This technology offers the means to begin
homology. For example, apoplast-localized defensins, MsDef1 development of durable disease-resistant crops for increased
385 DOI: 10.1021/acs.jafc.5b04543
J. Agric. Food Chem. 2016, 64, 383−393
Journal of Agricultural and Food Chemistry Perspective

Figure 3. Inhibition of F. graminearum PH-1 conidial germination and hyphal growth at different concentrations of MsDef1 (1.5 and 6 μM), MtDef4
(0.75 and 1.5 μM), and the MsDef1 variant, MsDef1-γ4, containing the γ-core motif of MtDef4 (1.5 and 3 μM). The γ-core motif of MtDef4 determines
the antifungal properties of MsDef1. Images were taken after 16 h of incubation of conidia with defensins. Bar = 50 μm. Hyperbranching of hyphae in the
presence of MsDef1 is indicated with a black arrow.

yields while substantially minimizing or eliminating the use of oxidation products, are being studied. A method termed the
environmentally harmful chemical fungicides. Successful deploy- “push−pull” utilizes companion crops to release semiochemicals,
ment of this technology will significantly contribute to food including these oxidized isoprenoids, to “push” away pests from
security and environmental sustainability. the food producing crop and to “pull” them into trap crops with

■
other uses.36 This is exemplified in a push−pull system for
SEMIOCHEMICALS AS NEW TARGETS FOR GM protecting cereals (e.g., maize and sorghum) produced for small-
CROPS holder sub-Saharan African farmers, who are not normally able to
purchase insecticides.37 Under this system, the trap crops
The main currently registered insecticides, fungicides, and
comprise valuable ruminant forage grasses, and the intercrops
herbicides comprise small lipophilic molecules (SLMs) often
comprise the cattle forage grass molasses grass (Melinis
developed from, or inspired by, natural product leads.33 For
minutiflora) or various species of the forage legumes in the
example, the recently introduced butenolide insecticides, typified
genus Desmodium, which release oxidized isoprenoids termed
by flupyradifurone, are structurally related to the plant secondary
homoterpenes, such as (E)-4,8-dimethyl-1,3,7-nonatriene (I).
metabolite stemofoline.34 These SLMs can also be obtained
directly from natural-based systems, particularly fermentation This approach is already being taken up by tens of thousands of
organisms (e.g., spinosad comprising spinosyns obtained from farmers in East Africa,37 thereby demonstrating the value of these
the yeast Saccharopolyspora spinosa). Thus, in nature there exist homoterpenes in insect pest management. Thus, the homo-
the genes for the biosynthesis of SLM insecticides, and other terpenes present potentially valuable new targets for using
pesticides, that could be used to furnish a new generation of GM defense semiochemicals in GM crops, and associated biosyn-
crops resistant to pests. GM crops expressing these SLMs as thesis genes are being characterized.
pesticides could have the same efficacy as current pesticides but For the new approaches described herein, nonconstitutive
with the added advantage that they could be delivered, as with expression of the associated genes represents another innovative
earlier GM pest-resistant crops, sustainably via the seed or other aspect. Thus, gene promotor sequences are employed that are
planting material.35 activated via use of a further group of SLMs active as defense
There are many natural SLMs showing promise as targets for elicitors. These include cis-jasmone, which, although related to
new GM pest-resistant crops, and these include compounds that the plant stress-induced and defense-related hormone jasmonic
because of instability and potential nontarget effects could be acid (JA), signals differently38 and elicits defense in a more
developed only by GM routes (e.g., the benzoxazinoids or consistent way39 and without phytotoxicity35 as is mostly found
hydroxamic acids).33 In addition to directly toxic MOA, GM with JA. cis-Jasmone can also elicit priming of defense in which
pest-resistant crops expressing SLMs that act through the effect is manifested once the insect attack occurs.40 New
sophisticated signaling mechanisms (e.g., involving pheromones elicitors are being characterized that, as with cis-jasmone, do not
and other semiochemicals (signals acting between species)) also require introduction to the plant by feeding damage, for example,
have great promise for emerging MOA.35 For example, the aphid from eggs of the pest41 and via mycorrhizal mycelial net-
alarm pheromone, (E)-β-farnesene, has a negative impact on works.42−45
aphid pests and increases foraging by antagonistic organisms By exploiting the approach of deploying semiochemicals via
such as aphid parasitoids. Genetic engineering in an elite wheat GM crops, a sustainable system for seed delivery of pest
variety has produced stable expression of highly pure (E)-β- management in food crops is created. Development of new MOA
farnesene, giving excellent results in the laboratory against cereal based on SLMs has great potential for controlling insect pests
aphids and in increasing foraging by braconid parasitoids of and providing alternatives for managing insect resistance. The
aphids. Field trial results33 are currently being analyzed. additional benefits of yield stability and reduced pesticide use will
In addition to pheromones, new targets for other semi- be important considerations for food security and agricultural
ochemicals, including a series of defense-related isoprenoid sustainability.
386 DOI: 10.1021/acs.jafc.5b04543
J. Agric. Food Chem. 2016, 64, 383−393
Journal of Agricultural and Food Chemistry Perspective

■ ENGINEERING ABIOTIC STRESS TOLERANCE IN

CROP PLANTS
for fresh water by allowing increased use of lower quality
(brackish) water.
Drought Tolerance and Water Use Efficiency Technol-
Fertilizer, water availability, and water quality are critical for crop
ogy. The technology being developed at Arcadia to address
productivity, and their availability directly affects food security in water deficit stress is based on the production of cytokinins under
both developed and developing nations. Salinity and water deficit stress conditions. This approach has been shown in tobacco51 to
stress alone account for >35% loss in agricultural yield potential, be effective in preserving yields under chronic deficit irrigation
and the efficiency of applied nitrogen fertilizers is ≤50% due to and at mitigating yield loss under extended periods of soil drying.
losses attributable to leaching, runoff, soil fixation, and gaseous The transgenic construct (pSARK-IPT) contains a maturation-
emissions.46 Working with commercial and humanitarian induced promoter (SARK) that controls the expression of
development partners, Arcadia has seen positive yield results in isopentenyltransferase (IPT), which is the rate-limiting enzyme
the field for technologies that improve nitrogen use efficiency in cytokinin biosynthesis. Plants typically respond to water stress
(NUE), water deficit stress tolerance, and salinity tolerance. by reducing transpiration. Initially this will induce stomatal
These technologies have also shown positive results in our first closing. Senescence and abscission of leaves for the recovery of
field tests with combinations of nitrogen and water deficit stress. nitrogen and photoassimilates and the reduction of canopy size
Nitrogen Use Efficiency. Engineering of plants to be more are typical adaptive responses that allow plants to set seed under
nitrogen efficient would both increase farmer productivity and prolonged or severe stress. However, the yield is greatly reduced.
decrease the environmental impact of nitrogen applications. The In crop plants, a severe yield reduction is considered crop failure.
NUE technology developed at Arcadia is based on modulating Better control over senescence initiation via cytokinin
the expression of an aminotransferase gene. Whereas the GS/ production provides protection against yield losses in pSARK-
GOGAT cycle is the major route of nitrogen (N) assimilation in IPT transgenic plants subjected to limiting water conditions.
plants, altering the expression of enzymes directly involved in this Combined Technologies. Crop plants are often affected by
cycle has not led to reproducible, field-demonstrated NUE. different abiotic stresses during a single growing season.
Aminotransferases are integral to N assimilation for the Therefore, combining NUE and drought- and salinity-tolerance
production of amino acids and N allocation in plants. Alanine technologies into a single construct is technically and strategically
aminotransferase enzymes catalyze the reversible formation of advantageous even without considering the potential synergies
alanine and 2-oxoglutarate from glutamate and pyruvate. between the technologies. Field trials of rice incorporating the
Increased NUE in transgenic plants expressing an alanine “triple-stack” technology, which includes NUE, water use
aminotransferase (AlaAT) from Hordeum vulgare under the efficiency (WUE), and salt tolerance (ST), have shown
control of a stress-inducible promoter from the Brassica napus substantial yield increases over conventional rice. In two years
turgor-responsive gene (btg26) was first demonstrated in of field trials, Arcadia’s triple-stack rice produced yield increases
canola.47 Arcadia’s NUE technology enables plants to absorb of 13−18% under various nitrogen application rates, 12−17%
and utilize nitrogen fertilizer much more efficiently than their under water stress conditions, and 15% under combined stress.
nontransgenic controls. This results in the same high yields as Parallel trials under salt stress in greenhouse conditions showed
conventional crops, while using half as much nitrogen fertilizer, yield increases as high as 42%. These results demonstrate that
these traits can work in combination to improve crop
or higher yields if using the same amount of fertilizer
performance under a number of stress conditions.
(unpublished results). In either case, less nitrogen escapes into
Both NUE alone and stacked technologies in the NERICA
the water and air. (New Rice for Africa, an interspecific hybrid rice developed
Salt Tolerance Technology. Arcadia’s salinity-tolerance specifically for dryland cultivation) rice background are being
technology is based on the overexpression of plant vacuolar Na+/ developed in collaboration with the African Agricultural
H+ antiporter(s) (NHXs).48,49 Vacuolar NHXs catalyze the Technology Foundation (AATF). Field trials have demonstrated
electroneutral exchange of cytoplasmic sodium (and potassium) NUE efficacy at The International Center for Tropical
with vacuolar protons. NHX overexpression promotes the Agriculture (CIAT) in Colombia, and trials are ongoing in
sequestering of sodium ions into the vacuoles of the cells, Ghana, Uganda, and Nigeria. Arcadia is developing similar trait
where it is not toxic and contributes favorably to the osmotic stacks in other crucial food and feed crops, including wheat and
balance of the cells and plant tissues. This strategy, which is based soybeans. Developing and developed nations will be affected by
on the characteristic high activity of vacuolar NHX activity the need to support a growing global population while utilizing
observed in salt-tolerant halophytes, promotes the tolerance of limited agricultural resources such as land, fertilizer, and water.
shoot tissues to sodium. There is also evidence that the With traits such as NUE, WUE, and ST, alone or in combination,
overexpression of NHX in roots promotes K+ homeostasis we expect a reduction in demand on these limited resources.
under saline conditions.50 It permits the growth and production This, combined with improvements in yield traits, pest
of seed under salinity stress levels that would otherwise have a resistances, and sustainable farming practices, will be a
negative impact on yield. The technology is applicable to a wide contributor in overall improved global food security.
range of crops, including maize, rice, soybeans, wheat, and
vegetables. Arcadia is also currently evaluating complementary
salinity tolerance genes for rice in collaboration with the United
■ GENETICALLY MODIFIED (GM) BANANAS
RESISTANT TO XANTHOMONAS WILT (BXW)
States Agency for International Development (USAID) and the DISEASE
Bangladesh Rice Research Institute (BRRI), with the aim of Banana and plantain (Musa sp.), the eighth most important
providing robust yields on cropland under chronic salinity stress. staple food crop in the tropics and subtropics, is grown in more
Arcadia’s salt-tolerance technology can improve farming than 140 countries, with an annual world production of around
efficiencies and reduce the need to expand agricultural activities 144 million tons.52 Approximately one-third of total bananas are
into new areas. In addition, this technology can reduce the need produced in Africa, mainly in the Great Lakes region of East
387 DOI: 10.1021/acs.jafc.5b04543
J. Agric. Food Chem. 2016, 64, 383−393
Journal of Agricultural and Food Chemistry Perspective

Africa, including Burundi, Rwanda, Democratic Republic of gene by PCR and Southern blot analysis and gene expression by
Congo, Uganda, Kenya, and Tanzania.52 However, banana qRT-PCR. The transgenic lines were evaluated for resistance
production is affected by many diseases, such as black Sigatoka, against Xcm using rapid in vitro bioassays and artificial
Fusarium wilt, banana Xanthomonas wilt (BXW), banana bunchy- inoculation of potted plants under glasshouse conditions. The
top virus (BBTV), and banana streak virus (BSV), and pests, transgenic banana plants conferred strong resistance to BXW, in
such as weevils and nematodes.53,54 both the in vitro assay and glasshouse screening test.71,72 The 65
BXW, caused by Xanthomonas campestris pv. musacearum resistant lines, selected on the basis of enhanced resistance to
(Xcm), is considered to be one of the most devastating diseases of BXW using potted plants in the glasshouse, the presence of low
banana and the biggest threat to banana production in the Great copies of the transgene, and detectable gene expression, were
Lakes region of eastern and central Africa.55 BXW was first further evaluated in a confined field trial at the National
reported in Ethiopia in Ensete species and then in banana.56,57 Agricultural Research Laboratory (NARL), Kawanda, Uganda.
Outside Ethiopia, BXW was first identified in Uganda in 200154 The majority of transgenic lines had significantly higher
and subsequently in the Democratic Republic of Congo,58 resistance in comparison to control nontransgenic plants.73
Rwanda,59 Kenya, Tanzania, and Burundi.60 Once BXW is Eleven of these transgenic lines (7 Hrap lines and 4 Pf lp lines)
established in fields, it is difficult to manage due to lack of were highly resistant, demonstrating 100% disease resistance for
availability of chemicals, biocontrol agents, or resistant two successive crops cycles (mother and ratoon) in comparison
varieties.55 Currently, the disease is managed by cultural to control nontransgenic plants. Approximately 85−93%
practices, including cutting and burying of infected plants, resistance with mother plants and 100% resistance with ratoon
restricting the movement of BXW-infected banana suckers plants was also observed in an additional five lines. The field trial
(planting materials) from affected fields to disease-free areas, results also confirmed the transfer of the disease resistance trait
debudding, and the use of sterilized farming tools. However, the from mother to progeny in several lines. Aside from enhanced
adoption of such practices has been inconsistent among farming resistance to BXW, the transgenic lines also showed flowering
communities as they are very labor intensive.61 and yields (bunch weight and fruit size) similar to those of
The lack of known genetic resistance in banana germplasm nontransgenic plants, indicating there were no observable
against Xcm, the difficulties associated with conventional unintended impacts of the transgenes on crop performance.73
breeding of this vegetatively propagated crop, and low adoption The best 10 lines were further planted in a second confined trial
of labor-intensive cultural practices favor biotechnological with more replicates to test the durability of disease resistance
approaches to develop BXW-resistant varieties. Transgenic and agronomic performance. These lines will be grown in
technology has opened new horizons in banana improvement, multilocation trials to test them in different environmental and
particularly for varieties that are not amenable to conventional climate conditions. It is well-known that pathogens can evolve
breeding. Due to lack of cross-fertile wild parents in many and single gene-based disease resistance can break down. To
banana-producing areas and the male and female sterility of most avoid this, we are stacking these two genes together in the same
edible cultivars and clonal propagation, there is a low risk of gene line to enhance the durability of the resistance trait.
flow, which makes a transgenic approach even more attractive. All transgenic plants are required to undergo thorough and
In the absence of known host plant resistance against Xcm in rigorous safety and risk assessments before commercialization.
banana germplasm, scientists at the International Institute of The HRAP and PFLP proteins are not listed as being potential
Tropical Agriculture (IITA) and the National Agriculture allergens in AllergenOnline, predicting that these proteins are
Research Organization (NARO)Uganda have been inves- safe for human consumption.73 These proteins are present in
tigating the potential of defense genes, hypersensitive response- several plant species, such as tobacco, Arabidopsis, rice, and
assisting protein (Hrap) and plant ferredoxin-like protein (Pf lp) vegetable crops such as pepper, which are even eaten raw as
from sweet pepper, for BXW disease resistance in banana. The salads. The banana transgenic lines will be analyzed for food and
HRAP is a plant protein that enhances the harpinPSS-mediated environmental safety in compliance with biosafety regulations
hypersensitive response (HR), a common plant defense before the varieties are released to farmers. Currently, we are
mechanism to protect plants against invading pathogens.62 studying environmental impacts, such as nontarget effects of
HRAP dissociates harpinPSS multimeric forms into dimers and disease-resistant bananas on soil microorganisms in the banana
monomers, which induces a stronger hypersensitive cell death rhizosphere.
(HCD) necrosis. There are studies demonstrating enhanced The transgenic bananas expressing Hrap and Pf lp may also
resistance against virulent pathogens in transgenic tobacco and provide resistance to other bacterial diseases, such as moko,
Arabidopsis overexpressing the Hrap gene.63,64 Similarly, the bugtok, or blood disease, which are affecting banana production
overexpression of sweet pepper Pf lp gene in transgenic tobacco, in Latin America, Caribbean, Philippines, and Indonesia. Bananas
tomato, orchids, calla lily, and rice has shown resistance against a are vitally important for sub-Saharan Africa, not only for food
broad range of bacterial pathogens such as Erwinia, Pseudomonas, security, but also as an important local cash crop for small-scale
Ralstonia, and Xanthomonas spp.65−69 The disease resistance farmers. Food security studies revealed that bananas constitute
provided by overexpression of the sweet pepper Pf lp gene in 30−60% of the daily per capita caloric intake in Uganda, Rwanda,
transgenic plants is reported to be due to induction of defense and Burundi.74 BXW-resistant varieties would boost the available
responses through enhanced production of active oxygen species arsenal to fight the BXW disease epidemic and save the
(AOS) and activation of the HR in resistant plants during livelihoods of African farmers, who depend on bananas as a
infection with bacterial pathogens.66,70 staple food crop.
The wilt-resistance genes, Pf lp and Hrap, were licensed by the
AATF on a royalty basis from the patent holder Academia Sinica
in Taiwan. IITA and NAROUganda have developed hundreds
■ VIRUS-RESISTANT CASSAVA FOR AFRICA (VIRCA)
Among the crops being grown by small-holder farmers in sub-
of transgenic lines of banana using Hrap and Pf lp genes. These Saharan Africa, cassava (Manihot esculenta Crantz), a root crop, is
lines were characterized for the presence and copy number of an important staple food crop. It is estimated to be a major source
388 DOI: 10.1021/acs.jafc.5b04543
J. Agric. Food Chem. 2016, 64, 383−393
Journal of Agricultural and Food Chemistry Perspective

Figure 4. Cassava brown streak disease (CBSD) resistance using RNAi technology in cassava: (left) non-transgenic control; (right) transgenic event
expressing the siRNA cassette.

of food and income for more than 250 million people in Africa VIRCA was initiated in 2005 and is currently in the second phase
and nearly a billion people globally.75,76 Cassava is a hardy and of funding for technology development and confined field testing
drought-tolerant crop that can be grown on marginal lands with for the release of improved varieties to small-holder farmers.81
very little input costs for farmers. Cassava is also an ideal food The primary focus of the project in this phase is to characterize
security crop for small-holder farmers because the tuberous roots and test the transgenic CBSD resistance lines developed using
can be stored in the soil for up to 3 years after planting without the RNAi technology. For CBSD resistance, an expression
deterioration in quality and can be harvested as needed.77,78 In cassette designed to generate siRNAs against the coat protein
addition to being a staple food crop, cassava roots and flour offer (CP) sequences of two viruses (CBSV and UCBSV) that cause
a huge potential for use in feed, starch, brewery, biofuel, and CBSD was inserted into the transgenic lines of farmer-preferred
other industrial applications. Despite substantial efforts being cassava varieties. Confined field trials, approved by the
made by agronomists and breeders for crop improvement, the appropriate national regulatory authorities, with the transgenic
yield of cassava in Africa remains very low compared to other lines are currently in progress for elite event selection in both
parts of the world and substantially lower than those of other Uganda and Kenya. To date, the results obtained from these trials
food crops grown with intensive crop management practices.75,79 demonstrate conclusively the efficacy of RNAi technology for
In addition to lack of resources available to small-holder sustained, season-long, and durable control of CBSD necrotic
farmers for input costs, biotic factors impose severe limitations root rot (Figure 4). The elite events selected from these trials will
on the yield and productivity of cassava. In particular, two viral be tested in multilocation trials in both countries to generate data
diseases, cassava mosaic disease (CMD) and cassava brown for full regulatory dossiers.
streak disease (CBSD), cause major yield losses.80,81 CMD is a The VIRCA project will provide the improved virus-resistant
foliar disease that has resulted in devastating yield losses in sub- varieties to small-holder farmers in Uganda and Kenya through
Saharan Africa. Recently, several tolerant varieties have been existing distribution channels and with no technology cost to the
developed through conventional breeding to combat CMD using farmer. However, approval from in-country regulatory agencies is
the source of resistance available within the cassava germplasm.
required before improved transgenic varieties can be distributed
CBSD affects cassava by causing brown necrotic lesions on roots,
to farmers. Following international guidelines, the VIRCA team
resulting in complete spoilage of edible roots and up to 100%
is working with national government regulatory authorities in
yield loss. CBSD is spreading in East Africa and is considered to
Kenya and Uganda to define the specific data requirements for
be a major threat to cassava production in this region.80 CBSD
has been recognized as one of the seven most dangerous crop food, feed, and environmental safety evaluation of virus-resistant
diseases in the world, capable of severely affecting global food cassava varieties for eventual general release. In Uganda and
security.82 CBSD is caused by two viruses, cassava brown streak Kenya, excellent infrastructure exists for confined field testing of
virus (CBSV) and Ugandan cassava brown streak virus transgenic events. Confined field trials are, therefore, progressing
(UCBSV), belonging to the Potyviridae family. CBSD shows smoothly in compliance with national regulatory authorities’
subtle and hardly noticeable foliar symptoms but results in guidelines and regulations. Each successful field trial generates
complete spoilage of edible roots. At present, for CBSD there is data for an eventual dossier for safety assessments that would
no reliable source of resistance available in cassava germplasm for enable approval of improved cassava varieties for small farmers to
use in conventional breeding programs. grow, resulting in increased amount of available food and
The VIRCA project is focused on developing pathogen-based income. Even so, the situation in each country may present a
RNAi technology to combat viral diseases for increasing yield challenge in the final stages of regulatory approvals. For example,
and production of cassava. Using this technology, researchers at in Kenya, the Biosafety Bill was passed in 2009, but there is no
the Institute for International Crop Improvement (IICI) at precedence for farm release of a GM crop. In Uganda, the
Danforth Center, USA, in collaboration with the National Crops Biosafety Bill has been advanced to the Ugandan Parliament and
Resources Research Institute (NaCRRI) in Uganda, the Kenyan is likely to be enacted soon. Although there is a very strong
Agricultural and Livestock Research Organization (KALRO), commitment and willingness on the part of technology
and the International Institute of Tropical Agriculture (IITA) in developers, partner organizations, and government agencies for
Kenya, are developing improved virus-resistant varieties of science-based approval and rapid release of virus-resistant cassava
cassava. VIRCA is a public and private sector partnership varieties, considering the current regulatory environment in both
supported by the Bill and Melinda Gates Foundation, the target countries, a much more concerted effort will be needed for
Howard Buffett Foundation, USAID, and the Monsanto Fund. general release of such varieties.
389 DOI: 10.1021/acs.jafc.5b04543
J. Agric. Food Chem. 2016, 64, 383−393
Journal of Agricultural and Food Chemistry Perspective

Sustainable agricultural production for food security is a global community, internal community, and future generations) for
issue, but the impact of food insecurity is likely to be more which appropriate indicators are included in the method.
pronounced in the developing world, especially in the sub- The AgBalance model was applied in a case study to assess the
Saharan African region. In this region, a large proportion of environmental and socioeconomic impacts of herbicide-tolerant
farmers are small land holders who are primarily dependent on and insect-resistant (Bacillus thuringiensis, Bt) GM maize
subsistence farming of crops, such as cassava, on marginal land varieties. In this case study, the production of non-GM maize
for food and income. The availability of virus-resistant cassava was compared to (1) herbicide-tolerant GM maize (specifically
varieties to small-holder farmers, therefore, will have a substantial tolerant to glyphosate (Roundup Ready, RR)); (2) GM maize
impact on improving the yield and productivity of cassava and containing both herbicide tolerance and insect resistance
will eventually play a major role in enhancing the food security (RR&Bt); and (3) a GM maize variety having multiple modes
and well-being of resource-poor farmers. of both herbicide tolerance and insect resistance (SmartStax).91

■
Results from this study have been described previously.91 Briefly,
USING LIFE CYCLE ASSESSMENT (LCA) TO ASSESS the LCA of this case study showed a positive effect from the GM
ENVIRONMENTAL AND SOCIOECONOMIC products in six of eight categories. These categories included land
IMPACTS OF GM CROPS use, soil impact, ecotoxicity potential, emission, energy
consumption, and resource consumption. Of the remaining
The benefits of the current agricultural biotechnology traits two categories, biodiversity and water use, positive results were
(specifically insect-resistant and herbicide-tolerant GM crops) observed for RR&Bt and SmartStax but not for RR. The negative
are well-documented. They have been shown to reduce the use of result for RR on biodiversity was potentially due to the decreased
pesticides, labor, and machinery and at the same time have weed abundance in the maize field and other indirect effects of
helped to increase yields or provide greater yield stability. the herbicide. The increased herbicide usage was also associated
Nevertheless, the potential contributions of agricultural bio- with increased water usage, thus causing a negative result for
technology to support sustainable development in agriculture water use.
remain controversial. A holistic and comprehensive framework More specifically, the case study showed that land use was
for the assessment of the environmental and socioeconomic reduced by up to 26% compared to non-GM maize due to higher
impacts of different production systems may help to bring yields, and soil erosion was decreased by 72% by the adoption of
transparency to this discussion. no-till cultivation practices, which are favored by herbicide-
LCA is a useful tool for quantitative sustainability assessment tolerant corn varieties. The positive effect on ecotoxicity
along value chains and across industry sectors, and it is potential was related to reduced intensity of insecticide use.
increasingly being used to assess the environmental impacts of The life cycle perspective showed that the production and use of
agriculture (see, for example, refs 83−85). LCA is based on a fertilizer was the main driver in the energy consumption,
“cradle-to-grave” approach, which begins with the gathering of resource consumption, and greenhouse gas emissions categories.
raw materials from the earth to create an input needed in the For example, energy consumption was dominated by the
production system (in the case of agriculture, for example, production of fertilizer, which constituted roughly ≥75% of all
fertilizer or crop protection products) and ends at the point when energy required in all four production systems. Similarly, the
all materials are returned to the earth (through emissions or emissions impact category was highly determined by emissions
disposal). LCA enables the estimation of the cumulative related to the production and use of nitrogen fertilizer. For
environmental impacts resulting from all stages in the product example, greenhouse gas emissions were dominated by 89% in
life cycle, often including impacts not considered in more the case of non-GM to up to 95% in the case of SmartStax by
traditional analyses (e.g., raw material extraction, resource production of fertilizer and field emissions from nitrification
depletion, energy consumption). By including the impacts processes in soils. This LCA showed that GM traits contributed
throughout the product life cycle, LCA provides a comprehen- to increased productivity and increased sustainability from an
sive view of the environmental aspects of the product or process environmental as well as socioeconomic perspective. In the
and a more accurate picture of the true environmental trade-offs. aggregated sustainability score across all three dimensions, the
An international framework for LCA exists and gives guidance for RR, RR&BT, and SmartStax products support approximately 7,
standardized procedures for assessment.86,87 15, and 17% better overall sustainability scores than the non-
On the basis of this framework, a holistic methodology called GMO alternative, respectively. Particularly, the RR&BT and
AgBalance has been developed that integrates the results of up to SmartStax products supported higher yield, reduced cost, and
69 indicators covering the environmental, economic, and social reduced environmental burden from insecticide use. Herbicide-
aspects of agricultural production.88 In addition to considering tolerant maize may also have a positive overall impact if the trait
energy use, emissions, ecotoxicity, and resource efficiency, other provides yield stability and no-till cultivation practices are used,
environmental indicators relevant to agriculture are also included which may reduce soil erosion and fuel use associated with field
(e.g., land use, water use, soil health, and biodiversity). In terms operations. Although the data used in this case study do not
of economic assessment, both production costs and economic represent all agricultural biotechnology traits, they highlight the
performance are taken into account, with the functional unit value of LCA for idenitifying the main drivers of agricultural
being defined relative to quantity and quality (e.g., 1 ton of maize sustainability and inform our understanding of how agricultural
grain). Production costs are grouped into variable and fixed costs biotechnology can complement a portfolio of agricultural tools
for maximizing sustainability and food security in the future.

■
and are quantified using an overall total cost of ownership for the
defined functional unit.89 Economic performance is assessed
using farm profitability as the central criterion for economic DISCUSSION
sustainability. The social assessment in AgBalance is based on the Total arable land that is devoted to agriculture is limited and in
UNEP-SETAC guidelines for social LCA of products90 and some regions of the world may be of poor quality for intensive
defines five stakeholder categories (farmer, consumer, local agricultural production. Responsible stewardship of environ-
390 DOI: 10.1021/acs.jafc.5b04543
J. Agric. Food Chem. 2016, 64, 383−393
Journal of Agricultural and Food Chemistry Perspective

mental resources, such as water, topsoil, renewable and (5) James, C. Global Status of Commercialized Biotech/GM Crops: 2014
nonrenewable energy, and nutrient inputs, is central to ; ISAAA Brief 49; International Service for the Acquisition of
sustainable agriculture. In the past, traditional breeding has Agribiotech Application: Ithaca, NY, USA, 2014.
been used to select for crop varieties with improved character- (6) U.S. Department of Agriculture, Economic Research Service
istics (e.g., increased yield, improved stress tolerance, enhanced (USDA-ERS). USDA Economic Research Service Adoption of
Genetically Engineered Crops in the U.S.: Recent Trends in GE
nutrition). However, the advent of modern biotechnology and
Adoption; http://www.ers.usda.gov/data-products/adoption-of-
the development of GM crops have enhanced the breeder’s genetically-engineered-crops-in-the-us/recent-trends-in-ge-adoption.
toolbox and allowed agriculture to move much more rapidly on a aspx (accessed Nov 6, 2015).
trajectory toward sustainability. These technologies have a broad (7) Embrapa Arroz e Feijão. Levantamento Sistemático da Produçaõ
and proven track record of improving yield and prosperity, with ́
Agricola - IBGE (1985−2013), modificado pela Embrapa Arroz e Feijão;
small-holder farmers in developing regions benefiting more than http://www.cnpaf.embrapa.br/socioeconomia/index.htm (accessed
those in developed regions.92 The innovations discussed here are May 15, 2015).
just a few examples of what is possible for improved food security (8) Melo, L. C.; Del Peloso, M. J.; Faria, J. C.; Yokoyama, M.; Rosaria,
and environmental stewardship. Although these new technolo- L.; Brondani, R. P. V.; Brondani, C.; de Faria, L. C. Controle Genético da
gies hold much promise, it is important to recognize that Reação do Feijoeiro Comum ao Vı ́rus do Mosaico Dourado; Boletim de
agricultural biotechnology represents only one set of tools for Pesquisa e Desenvolvimento: 2005; 15, pp 15−16.
improving agricultural sustainability and food security. An (9) Bonfim, K.; Faria, J. C.; Nogueira, E. O. P. L.; Mendes, É. A.;
integrated approach including precision agriculture for input Aragão, F. J. L. RNAi-mediated resistance to bean golden mosaic virus in
applications, conservation tillage, cover cropping, increased crop genetically engineered common bean (Phaseolus vulgaris). Mol. Plant−
Microbe Interact. 2007, 20, 717−726.
diversity, and other best management practices will all be
(10) Aragao, F. J. L.; Faria, J. C. First transgenic geminivirus-resistant
important for enhancing sustainability and managing agricul- plant in the field. Nat. Biotechnol. 2009, 27, 1086−1088.
ture’s environmental burden.93 (11) Faria, J. C.; Valdisser, P. A. M. R.; Nogueira, E. O. P. L.; Aragão, F.

■ AUTHOR INFORMATION
Corresponding Author
J. L. RNAi-based bean golden mosaic virus-resistant common bean
(Embrapa 5.1) shows simple inheritance for both transgene and disease
resistance. Plant Breed. 2014, 133, 649−653.
(12) Lay, F. T.; Anderson, M. A. Defensins − components of the innate
*(J.A.A.) E-mail: jennifer.anderson@pioneer.com. Phone: (515) immune system in plants. Curr. Protein Pept. Sci. 2005, 6, 85−101.
535-3730. (13) Vriens, K.; Cammue, B.; Thevissen, K. Antifungal plant defensins:
Funding mechanisms of action and production. Molecules 2014, 19, 12280−
12303.
L.T. received funding from the United States Agency for (14) Thomma, B.; Cammue, B.; Thevissen, K. Plant defensins. Planta
International Development (USAID) to support research on 2002, 216, 193−202.
BXW resistant bananas. J.P. received funding from Biotechnol- (15) Thomma, B. P. H. J.; Cammue, B. P. A.; Thevissen, K. Mode of
ogy and Biological Sciences Research Council (BBSRC) grants action of plant defensins suggests therapeutic potential. Curr. Drug
(including 2011−2016 PI, Grant BB/H017011/1, and 2013− Targets: Infect. Disord. 2003, 3, 1−8.
2017 PI, Grant BB/L001683/1). S.H. received funding from (16) Lacerda, A. F.; Vasconcelos, É. A.; Pelegrini, P. B.; Grossi de Sa, M.
USAID (Contract AEG-A-00-08-00009-00) and the African F. Antifungal defensins and their role in plant defense. Front. Microbiol.
Agricultural Technology Foundation (AATF, Contract AID- 2014, 5, 1−10.
OAA-A-14-00035). T.L.P.O.S. received funding from the (17) Sagaram, U. S.; Pandurangi, R.; Kaur, J.; Smith, T. J.; Shah, D. M.
Brazilian Agricultural Research Corporation (Embrapa), Brazil- Structure-activity determinants in antifungal plant defensins MsDef1
ian government. and MtDef4 with different modes of action against Fusarium
graminearum. PLoS One 2011, 6, e18550.
Notes (18) Hegedü s, N.; Marx, F. Antifungal proteins: more than
All authors participated in the drafting of this paper as individual antimicrobials? Fungal Biol. Rev. 2013, 26, 132−145.
experts in their fields, and the authors are solely responsible for (19) Thevissen, K.; Terras, F. R. G.; Broekaert, W. F. Permeabilization
the contents. Any views expressed in this paper are the views of of fungal membranes by plant defensins inhibits fungal growth. Appl.
the authors and do not necessarily represent the views of any Environ. Microbiol. 1999, 65, 5451−5458.
organization, institution, or government with which they are (20) Thevissen, K.; Ghazi, A.; De Samblanx, G. W.; Brownlee, C.;
affiliated or employed. Osborn, R. W.; Broekaert, W. F. Fungal membrane responses induced
by plant defensins and thionins. J. Biol. Chem. 1996, 271, 15018−15025.
The authors declare no competing financial interest.

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(21) Ramamoorthy, V.; Cahoon, E. B.; Li, J.; Thokala, M.; Minto, R. E.;
Shah, D. M. Glucosylceramide synthase is essential for alfalfa defensin-
REFERENCES mediated growth inhibition but not for pathogenicity of Fusarium
(1) How to feed a hungry world. Nature 2010, 466, 531−532.10.1038/ graminearum. Mol. Microbiol. 2007, 66, 771−786.
466531a (22) Thevissen, K.; Osborn, R. W.; Acland, D. P.; Broekaert, W. F.
(2) Tilman, D.; Balzer, C.; Hill, J.; Befort, B. L. Global food demand Specific binding sites for an antifungal plant defensin from dahlia
and the sustainable intensification of agriculture. Proc. Natl. Acad. Sci. U. (Dahlia merckii) on fungal cells are required for antifungal activity. Mol.
S. A. 2011, 108, 20260−20264. Plant−Microbe Interact. 2000, 13, 54−61.
(3) Food and Agriculture Organization of the United Nations (FAO). (23) van der Weerden, N. L.; Lay, F. T.; Anderson, M. A. The plant
Rome Declaration on World Food Security and World Food Summit Plan of defensin, NaD1, enters the cytoplasm of Fusarium oxysporum hyphae. J.
Action, Proceedings of the World Food Summit, Nov 13−17; Rome, Biol. Chem. 2008, 283, 14445−14452.
Italy, 1996. (24) Lobo, D. S.; Pereira, I. B.; Fragel-Madeira, L.; Medeiros, L. N.;
(4) Secretariat of the Convention on Biological Diversity. Article 2 Use Cabral, L. M.; Faria, J.; Bellio, M.; Campos, R. C.; Linden, R.;
of Terms. In Handbook of the Convention on Biological Diversity Including Kurtenbach, E. Antifungal Pisum sativum defensin 1 interacts with
its Cartagena Protocol on Biosafety, 3rd ed.; Montreal, Canada, 2005; Neurospora crassa cyclin F related to the cell cycle. Biochemistry 2007, 46,
ISBN 92-9225-011-6. 987−996.

391 DOI: 10.1021/acs.jafc.5b04543

J. Agric. Food Chem. 2016, 64, 383−393
Journal of Agricultural and Food Chemistry Perspective

(25) Sagaram, U. S.; El-Mounadi, K.; Buchko, G. W.; Berg, H. R.; Kaur, (44) Babikova, Z.; Gilbert, L.; Bruce, T.; Dewhirst, S. Y.; Pickett, J. A.;
J.; Pandurangi, R. S.; Smith, T. J.; Shah, D. M. Structural and functional Johnson, D. Arbuscular mycorrhizal fungi and aphids interact by
studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: changing host plant quality and volatile emission. Funct. Ecol. 2014, 28,
identification of an RGFRRR motif governing fungal cell entry. PLoS 375−385.
One 2013, 8, e82485. (45) Babikova, Z.; Johnson, D.; Bruce, T.; Pickett, J.; Gilbert, L. How
(26) Stotz, H. U.; Thomson, J.; Wang, Y. Plant defensins. Plant rapid is aphid-induced signal transfer between plants via common
Signaling Behav. 2009, 4, 1010−1012. mycelial networks? Commun. Integr. Biol. 2013, 6, e25904.
(27) Kaur, J.; Sagaram, U. S.; Shah, D. M. Can plant defensins be used (46) Baligar, V.; Fageria, N.; He, Z. Nutrient use efficiency in plants.
to engineer durable commercially useful fungal resistance in crop plants? Commun. Soil Sci. Plant Anal. 2001, 32, 921−950.
Fungal Biol. Rev. 2011, 25, 128−135. (47) Good, A. G.; Johnson, S. J.; De Pauw, M.; Carroll, R. T.; Savidov,
(28) Carvalho, A. d. O.; Gomes, V. M. Plant defensins − prospects for N.; Vidmar, J.; Lu, Z.; Taylor, G.; Stroeher, V. Engineering nitrogen use
the biological functions and biotechnological properties. Peptides 2009, efficiency with alanine aminotransferase. Can. J. Bot. 2007, 85, 252−262.
30, 1007−1020. (48) Apse, M. P.; Aharon, G. S.; Snedden, W. A.; Blumwald, E. Salt
(29) Carvalho, A. d. O.; Moreira Gomes, V. Plant defensins and tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in
defensin-like peptides − biological activities and biotechnological Arabidopsis. Science 1999, 285, 1256−1258.
applications. Curr. Pharm. Des. 2011, 17, 4270−4293. (49) Zhang, H.-X.; Hodson, J. N.; Williams, J. P.; Blumwald, E.
(30) Uma Shankar, S.; Jagdeep, K.; Shah, D. M. Antifungal plant Engineering salt-tolerant Brassica plants: characterization of yield and
defensins: structure-activity relationships, modes of action, and biotech seed oil quality in transgenic plants with increased vacuolar sodium
applications. In Small Wonders: Peptides for Disease Control; Rajasekaran, accumulation. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 12832−12836.
K., Cary, J. W., Jaynes, J. M., Montesinos, E., Eds.; ACS Symposium (50) Rodríguez-Rosales, M. P.; Jiang, X.; Gálvez, F. J.; Aranda, M. N.;
Series 1095; American Chemical Society: Washington, DC, USA, 2012; Cubero, B.; Venema, K. Overexpression of the tomato K+/H+ antiporter
pp 317−336. LeNHX2 confers salt tolerance by improving potassium compartmen-
(31) Kaur, J.; Thokala, M.; Robert-Seilaniantz, A.; Zhao, P.; Peyret, H.; talization. New Phytol. 2008, 179, 366−377.
Berg, H.; Pandey, S.; Jones, J.; Shah, D. M. Subcellular targeting of an (51) Rivero, R. M.; Kojima, M.; Gepstein, A.; Sakakibara, H.; Mittler,
evolutionarily conserved plant defensin MtDef4.2 determines the R.; Gepstein, S.; Blumwald, E. Delayed leaf senescence induces extreme
outcome of plant−pathogen interaction in transgenic Arabidopsis. Mol. drought tolerance in a flowering plant. Proc. Natl. Acad. Sci. U. S. A. 2007,
Plant Pathol. 2012, 13, 1032−1046. 104, 19631−19636.
(32) Ntui, V. O.; Azadi, P.; Thirukkumaran, G.; Khan, R. S.; Chin, D. (52) Food and Agriculture Organization of the United Nations (FAO).
P.; Nakamura, I.; Mii, M. Increased resistance to fusarium wilt in FAOSTAT agricultural data, 2012; http://faostat.fao.org/ (accessed
transgenic tobacco lines co-expressing chitinase and wasabi defensin Nov 5, 2015).
genes. Plant Pathol. 2011, 60, 221−231. (53) Jones, D. R. Diseases of Banana, Abacá and Enset; CABI
(33) Pickett, J. A.; Aradottír, G. I.; Birkett, M. A.; Bruce, T. J. A.;
Publishing: Wallingford, UK, 1999.
Hooper, A. M.; Midega, C. A. O.; Jones, H. D.; Matthes, M. C.; Napier, J. (54) Tushemereirwe, W.; Kangire, A.; Ssekiwoko, F.; Offord, L.;
A.; Pittchar, J. O.; Smart, L. E.; Woodcock, C. M.; Khan, Z. R. Delivering
Crozier, J.; Boa, E.; Rutherford, M.; Smith, J. First report of
sustainable crop protection systems via the seed: exploiting natural
Xanthomonas campestris pv. musacearum on banana in Uganda. Plant
constitutive and inducible defence pathways. Philos. Trans. R. Soc., B
Pathol. 2004, 53, 802−802.
2014, 369, 20120281.
(55) Tripathi, L.; Mwangi, M.; Abele, S.; Aritua, V.; Tushemereirwe,
(34) Jeschke, P.; Nauen, R.; Beck, M. E. Nicotinic acetylcholine
W. K.; Bandyopadhyay, R. Xanthomonas wilt: a threat to banana
receptor agonists: a milestone for modern crop protection. Angew.
production in East and Central Africa. Plant Dis. 2009, 93, 440−451.
Chem., Int. Ed. 2013, 52, 9464−9485.
(56) Yirgou, D.; Bradbury, J. A note on wilt of banana caused by the
(35) Birkett, M. A.; Pickett, J. A. Prospects of genetic engineering for
robust insect resistance. Curr. Opin. Plant Biol. 2014, 19, 59−67. enset wilt organism Xanthomonas musacearum. East Afr. Agric. For. J.
(36) Pickett, J. A.; Woodcock, C. M.; Midega, C. A. O.; Khan, Z. R. 1974, 40, 111−114.
Push−pull farming systems. Curr. Opin. Biotechnol. 2014, 26, 125−132. (57) Yirgou, D.; Bradbury, J. Bacterial wilt of enset (Ensete ventri-
(37) Khan, Z. R.; Midega, C. A. O.; Pittchar, J. O.; Murage, A. W.; cosum) incited by Xanthomonas musacearum sp. n. Phytopathology 1968,
Birkett, M. A.; Bruce, T. J. A.; Pickett, J. A. Achieving food security for 58, 111−112.
one million sub-Saharan African poor through push−pull innovation by (58) Ndungo, V.; Eden-Green, S.; Blomme, G.; Crozier, J.; Smith, J. J.
2020. Philos. Trans. R. Soc., B 2014, 369, 20120284. Presence of banana Xanthomonas wilt (Xanthomonas campestris pv.
(38) Matthes, M.; Bruce, T.; Chamberlain, K.; Pickett, J.; Napier, J. musacearum) in the Democratic Republic of Congo (DRC). Plant
Emerging roles in plant defense for cis-jasmone-induced cytochrome Pathol. 2006, 55, 294−294.
P450 CYP81D11. Plant Signaling Behav. 2011, 6, 563−565. (59) Reeder, R.; Muhinyuza, J.; Opolot, O.; Aritua, V.; Crozier, J.;
(39) Smart, L.; Martin, J.; Limpalaër, M.; Bruce, T. A.; Pickett, J. Smith, J. Presence of banana bacterial wilt (Xanthomonas campestris pv.
Responses of herbivore and predatory mites to tomato plants exposed to musacearum) in Rwanda. Plant Pathol. 2007, 56, 1038−1038.
jasmonic acid seed treatment. J. Chem. Ecol. 2013, 39, 1297−1300. (60) Carter, B. A.; Reeder, R.; Mgenzi, S. R.; Kinyua, Z. M.; Mbaka, J.
(40) Oluwafemi, S.; Dewhirst, S. Y.; Veyrat, N.; Powers, S.; Bruce, T. J. N.; Doyle, K.; Nakato, V.; Mwangi, M.; Beed, F.; Aritua, V.; Lewis Ivey,
A.; Caulfield, J. C.; Pickett, J. A.; Birkett, M. A. Priming of production in M. L.; Miller, S. A.; Smith, J. J. Identification of Xanthomonas vasicola
maize of volatile organic defence compounds by the natural plant (formerly X. campestris pv. musacearum), causative organism of banana
activator cis-jasmone. PLoS One 2013, 8, e62299. xanthomonas wilt, in Tanzania, Kenya and Burundi. Plant Pathol. 2010,
(41) Tamiru, A.; Bruce, T. J.; Midega, C. A.; Woodcock, C. M.; Birkett, 59, 403−403.
M. A.; Pickett, J. A.; Khan, Z. R. Oviposition induced volatile emissions (61) Kagezi, G.; Kangire, A.; Tushemereirwe, W.; Bagamba, F.;
from African smallholder farmers’ maize varieties. J. Chem. Ecol. 2012, Kikulwe, E.; Gold, C.; Ragama, P.; Kubiriba, J. Banana bacterial wilt
38, 231−234. incidence in Uganda. Afr. Crop Sci. J. 2006, 14, 83−91.
(42) Babikova, Z.; Gilbert, L.; Bruce, T. J. A.; Birkett, M.; Caulfield, J. (62) Chen, C.; Lin, H.; Ger, M.; Chow, D.; Feng, T. The cloning and
C.; Woodcock, C.; Pickett, J. A.; Johnson, D. Underground signals characterization of a hypersensitive response assisting protein that may
carried through common mycelial networks warn neighbouring plants of be associated with the harpin-mediated hypersensitive response. Plant
aphid attack. Ecol. Lett. 2013, 16, 835−843. Mol. Biol. 2000, 43, 429−438.
(43) Babikova, Z.; Johnson, D.; Bruce, T.; Pickett, J.; Gilbert, L. (63) Ger, M.-J.; Chen, C.-H.; Hwang, S.-Y.; Huang, H.-E.; Podile, A. R.;
Underground allies: how and why do mycelial networks help plants Dayakar, B. V.; Feng, T.-Y. Constitutive expression of hrap gene in
defend themselves? BioEssays 2014, 36, 21−26. transgenic tobacco plant enhances resistance against virulent bacterial

392 DOI: 10.1021/acs.jafc.5b04543

J. Agric. Food Chem. 2016, 64, 383−393
 Journal of Agricultural and Food Chemistry Perspective

pathogens by induction of a hypersensitive response. Mol. Plant− (81) Taylor, N. J.; Halsey, M.; Gaitán-Solís, E.; Anderson, P.; Gichuki,
Microbe Interact. 2002, 15, 764−773. S.; Miano, D.; Bua, A.; Alicai, T.; Fauquet, C. M. The VIRCA project.
(64) Pandey, A.-K.; Ger, M.-J.; Huang, H.-E.; Yip, M.-K.; Zeng, J.; GM Crops Food 2012, 3, 93−103.
Feng, T.-Y. Expression of the hypersensitive response-assisting protein (82) Pennisi, E. Armed and dangerous. Science 2010, 327, 804−805.
in Arabidopsis results in harpin-dependent hypersensitive cell death in (83) Brentrup, F.; Kusters, J.; Kuhlmann, H.; Lammel, J. Environ-
response to Erwinia carotovora. Plant Mol. Biol. 2005, 59, 771−780. mental impact assessment of agricultural production systems using the
(65) Huang, H.-E.; Ger, M.-J.; Chen, C.-Y.; Pandy, A.-K.; Yip, M.-K.; life cycle assessment methodology − I. Theoretical concept of a LCA
Chou, H.-W.; Feng, T.-Y. Disease resistance to bacterial pathogens method tailored to crop production. Eur. J. Agron. 2004, 20, 247−264.
affected by the amount of ferredoxin-I protein in plants. Mol. Plant (84) Hayashi, K.; Gaillard, G.; Nemecek, T. Life cycle assessment of
Pathol. 2007, 8, 129−137. agricultural production systems: current issues and future perspectives.
(66) Huang, S.; Chen, C.; Lin, H.; Ger, M.; Chen, Z.; Feng, T. A In Good Agricultural Practice (GAP) in Asia and Oceania; Hu, S. H.,
hypersensitive response was induced by virulent bacteria in transgenic Bejosano-Gloria, C., Eds.; Food and Fertilizer Technology Center:
Taipei, Taiwan, 2007; pp 98−110.
tobacco plants overexpressing a plant ferredoxin-like protein (PFLP).
(85) Sonesson, U.; Berlin, J.; Ziegler, F. Environmental Assessment and
Physiol. Mol. Plant Pathol. 2004, 64, 103−110.
Management in the Food Industry: Life Cycle Assessment and Related
(67) Liau, C.-H.; Lu, J.-C.; Prasad, V.; Hsiao, H.-H.; You, S.-J.; Lee, J.-
Approaches; Woodhead Publishing: Cambridge, UK, 2010.
T.; Yang, N.-S.; Huang, H.-E.; Feng, T.-Y.; Chen, W.-H. The sweet (86) International Organization for Standardization (ISO). Environ-
pepper ferredoxin-like protein (pf lp) conferred resistance against soft mental Management  Life Cycle Assessment  Principles and Framework
rot disease in Oncidium orchid. Transgenic Res. 2003, 12, 329−336. (ISO 14040:2006); ISO: Geneva, Switzerland, 2006.
(68) Tang, K.; Sun, X.; Hu, Q.; Wu, A.; Lin, C.-H.; Lin, H.-J.; Twyman, (87) International Organization for Standardization (ISO). Environ-
R. M.; Christou, P.; Feng, T. Transgenic rice plants expressing the mental Management  Life Cycle Assessment  Requirements and
ferredoxin-like protein (AP1) from sweet pepper show enhanced Guidelines (ISO 14044:2006); ISO: Geneva, Switzerland, 2006.
resistance to Xanthomonas oryzae pv.oryzae. Plant Sci. 2001, 160, 1035− (88) Frank, M.; Schöneboom, J.; Gipmans, M.; Saling, P. Holistic
1042. sustainability assessment of winter oilseed rape production using the
(69) Yip, M.-K.; Huang, H.-E.; Ger, M.-J.; Chiu, S.-H.; Tsai, Y.-C.; Lin, AgBalance method−an example of ‘sustainable intensification’. In
C.-I.; Feng, T.-Y. Production of soft rot resistant calla lily by expressing a Proceedings of the 8th International Conference on Life Cycle Assessment in
ferredoxin-like protein gene (pf lp) in transgenic plants. Plant Cell Rep. the Agri-Food Sector; Corson, M. S., van der Werf, H. M. G., Eds.; INRA:
2007, 26, 449−457. Rennes, France; 2012; pp 58−64.
(70) Dayakar, B. V.; Lin, H.-J.; Chen, C.-H.; Ger, M.-J.; Lee, B.-H.; Pai, (89) Kicherer, A.; Schaltegger, S.; Tschochohei, H.; Ferreira Pozo, B.
C.-H.; Chow, D.; Huang, H.-E.; Hwang, S.-Y.; Chung, M.-C. Ferredoxin Combining life cycle assessment and life cycle costs via normalization.
from sweet pepper (Capsicum annuum L.) intensifying harpinpss- Int. J. Life Cycle Assess. 2007, 12, 537−543.
mediated hypersensitive response shows an enhanced production of (90) United Nations Environment Programme. UNEP Guidelines for
active oxygen species (AOS). Plant Mol. Biol. 2003, 51, 913−924. Social Life Cycle Assessment of Products, 2009; available at http://
(71) Tripathi, L.; Mwaka, H.; Tripathi, J. N.; Tushemereirwe, W. K. www.unep.fr/shared/publications/pdf/DTIx1164xPA-guidelines_
Expression of sweet pepper Hrap gene in banana enhances resistance to sLCA.pdf (accessed June 2, 2015).
Xanthomonas campestris pv. musacearum. Mol. Plant Pathol. 2010, 11, (91) Gipmans, M.; Schoeneboom, J.; Klein, D.; Bihlmeyer, D.; Saling,
721−731. P. Assessing the socio-economic and environmental impact of GMO
(72) Namukwaya, B.; Tripathi, L.; Tripathi, J.; Arinaitwe, G.; Mukasa, corn varieties and consequential changes in farm management practices.
In Proceedings of the 9th International Conference on Life Cycle Assessment
S.; Tushemereirwe, W. Transgenic banana expressing Pflp gene confers
in the Agri-Food Sector; Schenck, R., Huizenga, D., Eds.; ACLCA:
enhanced resistance to Xanthomonas wilt disease. Transgenic Res. 2012,
Vashon, WA, USA, 2014: pp 456−465.
21, 855−865. (92) Klümper, W.; Qaim, M. A meta-analysis of the impacts of
(73) Tripathi, L.; Tripathi, J. N.; Kiggundu, A.; Korie, S.; Shotkoski, F.; genetically modified crops. PLoS One 2014, 9, e111629.
Tushemereirwe, W. K. Field trial of Xanthomonas wilt disease-resistant (93) National Research Council (NRC). Toward Sustainable
bananas in East Africa. Nat. Biotechnol. 2014, 32, 868−870. Agricultural Systems in the 21st Century; The National Academies
(74) Abele, S.; Twine, E.; Legg, C. Food Security in Eastern Africa and Press: Washington, DC, USA, 2010; pp 3−14.
the great lakes. Crop Crisis Control Project final report, 2007; available
at http://www.crs.org/sites/default/files/tools-research/c3p-food-
security-in-eastern-africa-and-the-great-lakes-region.pdf (accessed Nov
4, 2015).
(75) OECD. Consensus Document on Compositional Considerations for
New Varieties of Cassava (Manihot esculenta Crantz): Key Food and Feed
Nutrients, Anti-nutrients, Toxicants and Allergens; Organization for
Economic Cooperation and Development: Paris, France, 2009.
(76) Gbadegesin, M. A.; Olaiya, C. O.; Beeching, J. R. African cassava:
biotechnology and molecular breeding to the rescue. Br. Biotechnol. J.
2013, 3, 305−317.
(77) Halsey, M. E.; Olsen, K. M.; Taylor, N. J.; Chavarriaga-Aguirre, P.
Reproductive biology of cassava (Manihot esculenta Crantz) and
isolation of experimental field trials. Crop Sci. 2008, 48, 49−58.
(78) OECD. Consensus Document on the Biology of Cassava (Manihot
esculenta Crantz); Organization for Economic Cooperation and
Development: Paris, France, 2014.
(79) Wedding, K.; Tuttle, J. N. Pathways to Productivity: the Role of
GMOs for Food Security in Kenya, Tanzania, and Uganda; CSIS Global
Food Security Project; Rowman & Littlefield: New York, 2013.
(80) Alicai, T.; Omongo, C.; Maruthi, M.; Hillocks, R.; Baguma, Y.;
Kawuki, R.; Bua, A.; Otim-Nape, G.; Colvin, J. Re-emergence of cassava
brown streak disease in Uganda. Plant Dis. 2007, 91, 24−29.

393 DOI: 10.1021/acs.jafc.5b04543

J. Agric. Food Chem. 2016, 64, 383−393

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